Prognostic and diagnostic value of circulating IGFBP2 in pancreatic cancer

Abstract Insulin-like growth factor binding protein 2 (IGFBP2) is overexpressed in tumor tissues of several malignancies, including pancreatic cancer. Because of its role in tumor progression, IGFBP2 has been investigated as a tumor biomarker. However, little is known about its utility in pancreatic cancer. Plasma IGFBP2 levels were determined using enzyme-linked immunosorbent assay in 75 patients with pancreatic ductal adenocarcinoma (PDAC), 73 matched healthy controls, and 17 chronic pancreatitis patients. Our results showed that the plasma IGFPB2 level was significantly higher in PDAC patients than in patients with chronic pancreatitis and healthy controls. At a cut-off value of 333.9 ng/mL, the specificity and sensitivity were 78.08 and 65.33%, respectively. IGFBP2 level alone did not outperform carbohydrate antigen 19-9 (CA19-9) in diagnostic accuracy, but it successfully identified 9 out of 24 PDAC patients who were misidentified by CA19-9. The combination of IGFBP2 and CA19-9 was more accurate in the detection of PDAC than CA19-9 alone. IGFBP2 was more accurate than the other in discriminating between chronic pancreatitis and PDAC. Plasma IGFBP2, rather than CA19-9, was higher in the new-onset diabetes, lymph node involvement, and distant metastasis subgroups. IGFBP2 level was notably higher in stage IV cases than in stage I/II or stage III disease. However, CA19-9 did not show a difference between stages. After adjusting for lymph node involvement and distant metastasis, plasma IGFBP2 was identified as an independent prognostic marker for PDAC. The median survival time for patients with an IGFBP2 level ≥333.9 ng/mL was significantly shorter than that for patients with an IGFBP2 level <333.9 ng/mL. Marked elevation of plasma IGFBP2 in PDAC is associated with poorer survival. IGFBP2 may be considered as a supplementary biomarker for the diagnosis and prognostic prediction in Chinese pancreatic cancer patients.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy worldwide.The incidence is increasing annually, while the 5-year overall survival rate remains less than 10% [1].Despite developments in surgical techniques and adjuvant chemotherapy, pancreatic cancer has shown a slow improvement in survival rates [2].The main reason for poor survival is late diagnosis.
Carbohydrate antigen 19-9 (CA19-9) is commonly used as a conventional biomarker for PDAC, but its universal application is limited for several reasons, including falsenegative results in 10% of cases with the Lewis-negative genotype as well as false-positive results in obstructive jaundice, chronic pancreatitis, and other malignancies.Although risk factors for PDAC such as diabetes mellitus and smoking, have been claimed, doctors are reluctant to use CA19-9 for screening the risk category because it is less effective [3].
The insulin-like growth factor-binding proteins (IGFBPs) are a family of proteins that bind to and serve as carriers of IGFs, prolonging the IGF half-life in the circulation and modulating local IGF concentrations and activities [4].IGFBP2 is the second most abundant circulating IGFBPs.IGFBP2 has shown diagnostic and prognostic potential in glioma [5], prostate cancer, and ovarian cancer [6].IGFBP2 was first discovered in the proteomic studies to be elevated of pancreatic juice from PDAC patients [7].Later, it was found to be elevated in PDAC tumor samples, but not in chronic pancreatitis tissue [8].In a randomized phase 2 trial, the plasma IGFBP2 level was found to be correlated with the response to treatment with gemcitabine and the IGF1 receptor inhibitor ganitumab in metastatic pancreatic cancer [9].
Kendrick et al. first discussed using plasma IGFBP2 and methoselin as a panel of potential diagnostic biomarkers in a US cohort of PDAC patients [10].Nevertheless, it is still not clear if plasma IGFBP2 level correlates with primary clinical variables and survival.Our purpose was to investigate plasma IGFBP2 levels in PDAC patients, chronic pancreatitis patients, and matched healthy controls in a Chinese cohort.Besides validating that plasma IGFBP2 level was significantly elevated in PDAC patients, we also showed important correlations between plasma IGFBP2 and certain clinical parameters.Finally, we showed that high levels of plasma IGFBP2 was correlated with poor survival in Chinese PDAC patients.

Study subjects
A total of 75 patients with newly diagnosed PDAC between 2009 and 2012 at Tianjin Cancer Hospital, Tianjin, China, were randomly selected into this retrospective study as the PDAC patient group.PDAC was confirmed in these patients through histological diagnosis on fine-needle biopsy specimens or immunohistochemical testing on surgical samples.We also included 17 patients with chronic pancreatitis (ChPT) who were admitted to the outpatient pancreatic cancer clinic of Tianjin Cancer Hospital for evaluation of a "pancreatic mass" identified at another institution.Another 73 persons without PDAC or ChPT were selected as age-and sex-matched healthy controls from among the individuals who visited Tianjin Cancer Hospital for cancer screening.Exclusion criteria included patients with other malignancies diagnosed or treated within the last 5 years.The study was approved by the research ethics committees of Tianjin Cancer Hospital, and written informed consent was obtained from each patient.

Blood sample collection and processing
Peripheral blood samples were collected with either heparin or ethylenediaminetetraacetic acid (EDTA) as an anti-coagulation agent.Plasma was isolated by subjecting the samples to centrifugation at 1,500g for 5 min at room temperature.A pilot study on three healthy controls showed no significant difference in IGFBP2 levels in plasma specimens obtained with either heparin or EDTA from the same person.Plasma samples were frozen and maintained at −80°C until the assays were conducted.

Enzyme-linked immunosorbent assay (ELISA) for IGFBP2
Plasma IGFBP2 was measured by an ELISA kit (R&D Systems, Minneapolis, USA), following the manufacturer's instructions.

Statistical analysis
Data were analyzed using IBM SPSS version 22 (SPSS Inc., Chicago, IL, USA) for Windows and GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA).Values of IGFBP2 were expressed as mean ± standard deviation (SD).Demographic data were analyzed by the chi-square test and continuous data were compared with an independent-sample Student's t-test.We compared the differences in plasma levels of IGFBP2 between cases and controls using the nonparametric Mann-Whitney U test and evaluated the relationship between IGFBP2 levels and clinical parameters through univariate linear regression analysis.We constructed survival curves according to plasma levels of IGFBP2 and CA19-9 using the Kaplan-Meier method and compared the median survival times by the log-rank test.
Hazard ratios (HRs) and 95% confidence intervals (CIs) for survival were estimated by multivariate Cox proportional hazard regression analysis, adjusting for lymph node involvement and distant metastasis.All P values were two-tailed, with 0.05 specified as the threshold for statistical significance.

Plasma IGFBP2 levels in patients with pancreatic cancer, chronic pancreatitis, and healthy controls
To assess the differences in IGFBP2 levels among patients with PDAC or ChPT and healthy controls, plasma IGFBP2 levels were determined blindly by ELISA.The mean plasma concentration of IGFBP2 in PDAC patients (435.7 ± 234.2 ng/mL) was significantly higher than that of either ChPT patients (233.0 ± 59.80 ng/mL, P = 0.0007) or healthy controls (289.9 ± 148.2 ng/mL, P < 0.0001; Figure 1a).However, no difference was observed between ChPT patients and healthy controls (P = 0.1254).In an analysis of these data by sex and age, IGFBP2 level was still higher in patients with PDAC than those with ChPT or healthy controls in each group (all P < 0.001; Figure 1b).Moreover, no obvious difference was found between healthy controls and nine ChPT patients in different sex or age groups.These results demonstrate that circulating IGFBP2 values were significantly higher in patients with PDAC, regardless of age or sex.

Specificity and sensitivity of plasma IGFBP2 as a diagnostic biomarker for pancreatic cancer
From the receiver operating characteristic (ROC) curve, we determined the specificity and sensitivity of IGFBP2 for diagnosis.At a cut-off value of 333.9 ng/mL, the specificity and sensitivity of IGFBP2 were 78.08 and 65.33%, respectively.The area under the ROC curve (AUC) of IGFBP2 was 0.727 (Figure 2a).The 95% specificity threshold of IGFBP2 yielded a sensitivity of 38.6%.CA 19-9, the most widely used biomarker for PDAC, yielded an AUC of 0.893 in our cohort.At the widely used clinical threshold of 37 U/mL, CA 19-9 produced a sensitivity of 73% and a specificity of 97% in this cohort.The 95% specificity threshold of CA 19-9 yielded a sensitivity of 75%.Although the diagnostic power of IGFBP2 alone was not as great as that of CA 19-9, IGFBP2 at its 95% specificity concentration (494 ng/mL) correctly  Value of IGFBP2 in pancreatic cancer  3 identified 9 out of the 24 cases misidentified by CA 19-9 at the 37 U/mL threshold.Furthermore, the AUC of the combination of IGFBP2 and CA19-9 reached 0.921.ROC curves were generated to compare the utility of plasma IGFBP2 levels in differentiating PDAC from ChPT (Figure 2b).IGFBP2 outperformed CA19-9 in diagnostic accuracy, with AUCs of 0.817 and 0.779, respectively (P < 0.001).The combination of IGFBP2 and CA19-9 yielded an AUC of 0.918 in differentiating PDAC from ChPT.

Plasma IGFBP2 in subgroups of pancreatic cancer patients
Plasma IGFBP2 levels in various PDAC patient subgroups were analyzed to determine the correlation between IGFBP2 and clinical parameters (Table 2; Figure 3).No difference was identified in the sex, age, or tumor size subgroups.
Patients with long-term diabetes had a relatively lower IGFBP2 than those without diabetes (336.4 ± 205.3 ng/mL vs 442.6 ± 176.2, P = 0.0359), while patients with new-onset diabetes exhibited a significantly higher IGFBP2 (585.4 ± 317.2 ng/mL) than patients without diabetes (P = 0.0466).CA19-9 levels did not differ significantly between the diabetes subgroups (Table 2; Figure 3e).IGFBP2 was elevated in patients with lymph node involvement (509.1 ± 271.3 ng/mL) and in those with distant metastasis (583.4 ± 258.1 ng/mL) compared to their respective control groups.However, CA19-9 does not show any difference between these groups (Figure 3f and g).Patients with stage I/II PDAC (388.3 ± 155.7 ng/mL, P = 0.0013) and those with stage III PDAC (364.9 ± 248.4 ng/mL, P = 0.0066) showed significantly lower IGFBP2 values than patients with stage IV PDAC (583.4 ± 258.1 ng/mL).This suggests that IGFBP2 levels increase as the disease progresses to an advanced stage.CA19-9 levels did not differ according to disease stage (Figure 3h).
In multivariate analysis, a plasma IGFBP2 level greater than the cut-off value of 333.9 ng/mL was found to be an independent predictor of survival (HR = 2.353, 95% CI 1.344-4.117,P = 0.003) after adjusting for lymph node involvement and distant metastasis.Distant metastasis was    identified as another independent factor.The results of the univariate and multivariate analyses are summarized in Table 3.The median survival time for patients with an IGFBP2 level ≥333.9 ng/mL was significantly shorter than that for patients with an IGFBP2 level <333.9 ng/mL (8 months vs 11 months, P < 0.001; Figure 4).No significant median survival was shown between patients grouped by CA19-9 cutoff value at 37 U/mL (P = 0.470).

Discussion
Our study demonstrated that the plasma IGFBP2 level was significantly higher in patients with PDAC than in patients with ChPT or healthy controls in a Chinese cohort, which is consistent with a recent single-center report from the United States [10].While IGFBP2 alone did not outperform CA19-9 in diagnostic power, as determined by sensitivity and specificity, IGFBP2 accurately identified more than one third of cases (9 out of 24) misidentified by CA19-9 in our cohort.
Rather than replacing CA 19-9 as a biomarker for PDAC, IGFBP2 may be an ideal complementary marker.The performance of CA19-9 in discriminating PDAC patients from healthy subjects is variable in the clinic, with some studies reporting barely acceptable AUC values of 0.83-0.84[11].In this study, we observed a very strong performance from CA19-9 in differentiating patients with PDAC from healthy controls (AUC = 0.893).The combination of IGFBP2 and CA19-9 was even stronger (AUC = 0.921).These data indicate the clinical potential of combining IGFBP2 and CA19-9 in the diagnosis of PDAC.Considering that PDAC is a heterogeneous tumor, it is reasonable to combine markers together rather than using a single one for diagnosis.
Chronic pancreatitis, especially autoimmune pancreatitis, is a risk factor for PDAC and sometimes is misdiagnosed as a malignant lesion.However, the treatments for pancreatitis and pancreatic cancer are absolutely different.Our findings show that plasma IGFBP2 was elevated exclusively in PDAC and not in ChPT or healthy controls, which is consistent with the findings of a previous study in tissue samples [12].From the ROC curves, we also demonstrated that IGFBP2 (AUC = 0.817) was a better marker than CA19-9 (AUC = 0.779) in discrimination of PDAC from ChPT.The combination of IGFBP2 and CA19-9 even more powerfully discriminated PDAC from ChPT (AUC = 0.918).
The relationship between diabetes mellitus and PDAC has long been recognized.Most cases of pancreatic cancerassociated diabetes are of new onset, defined as onset 2 years or less preceding the diagnosis of cancer [13].The ability to differentiate between pancreatic cancer-associated diabetes from the more common type 2 diabetes by a serological biomarker may be a great strategy for screening people at high risk of having asymptomatic pancreatic cancer.
In our analysis, the expression of IGFBP2 differed between patients with and without diabetes [14].As far as we know, our study is the first to compare the level of IGFBP2 in PDAC patients stratified by their diabetes status.In our cohort, PDAC patients with a long history of diabetes [13] exhibited lower plasma IGFBP2 levels than those without diabetes history.This result is not surprising, because previous studies have shown that IGFBP2 has an anti-diabetes effect [15] and is expressed at lower levels in patients with diabetes than in people without diabetes [16].Interestingly, we observed a remarkable aberrantly higher level of IGFBP2 in PDAC patients with new-onset diabetes than in PDAC patients with long-term diabetes or no diabetes.One possible explanation is that pancreatic cancer cells may secrete more IGFBP2 that cannot regulate the cancer-induced hyperglycemia.The data suggests that IGFBP2 has the potential to distinguish between the two types of diabetes in PDAC.However, one shortcoming of our study is the lack of diabetes status for the healthy controls; with that information, we could have determined the potential of IGFBP2 as an effective biomarker for PDAC screening in the diabetes risk group.
Our findings indicate that plasma IGFBP2 was elevated to the greatest extent in PDAC patients with either lymph node involvement or distant metastasis and that this elevation was associated with shorter overall survival of these patients that supports similar findings in other cancers, such as ovarian cancer [17], colorectal cancer [18], and prostate cancer [19].Univariate and multivariate survival analyses results suggest that plasma IGFBP2 level is an independent prognostic factor when adjusted for diabetes status, lymph nodes involvement, and distant metastasis.
It is interesting that our view on the role of IGFBP2 as a predictor of patient survival in PDAC differs from that of Kendrick et al. [10].One possible explanation for the divergence is the heterogeneity of the cohorts.Our univariate analysis considered absolutely different factors than theirs, except for IGFBP2, as predictors of survival.After adjustment by those different factors, it is possible to reach the opposite conclusion on IGFBP2's utility in predicting survival.Different ethnic groups may be another reason for the disagreement between our conclusion and that of Kendrick et al.
Taken together, our findings suggest that the plasma IGFBP2 level is significantly elevated in PDAC and that this elevation and its extent are related to tumor progression, glucose metabolism, and prognosis.Further evaluation of IGFBP2 as a biomarker in PDAC will require larger patient cohorts with a greater variation in disease stage.Specifically, more patients with early stage PDAC (i.e., PanIn) should be included to determine the early diagnostic value of IGFBP2.Immunohistochemical data could be adopted to investigate the correlation between systemic and localized IGFBP2 levels.More mechanistic studies are imperative to show how IGFBP2 is involved in these referred processes in PDAC.The integrin/ ILK/NF-κB [20], IGF1R/PI3K/AKT [21], and Wnt/β-catenin [22] pathways, as well as certain micro-RNAs have been proven to regulate or be regulated by IGFBP2 in a variety of malignant tumors.Characterization of IGFBP2-mediated pathways in pancreatic cancer cells may provide molecular insights into the progression of pancreatic cancer and suggest potential therapeutic targets for pancreatic cancer patients with elevated IGFBP2.In conclusions, this randomized, retrospective study has demonstrated that plasma IGFBP2 is significantly elevated in Chinese PDAC patients compared to ChPT or healthy controls.IGFBP2 is a complementary biomarker for PDAC because the combination of it with CA19-9 is more accurate in diagnosis than using CA19-9 alone.Higher plasma IGFBP2 is correlated with the new onset diabetes, lymph node involvement, distant metastasis, and advanced stage.Our findings also assert that elevated plasma IGFBP2 is an independent prognostic marker for PDAC.To further illustrate the clinic value of IGFBP2 in PDAC, a larger cohort with more early-stage patients and greater clinicopathological variation is necessary for future study.
Funding information: This project was partially supported by grants from the National Natural Science Foundation of China (Grant no.82072657).
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and consented to its submission to the journal, reviewed all the results and approved the final version of the manuscript.Jie Xu designed the experiments, Yuning Song and Bodong Zhou carried them out.Shuai Yuan developed the statistical analysis work.Song Gao prepared the manuscript with contributions from all co-authors.

Figure 1 :
Figure 1: Plasma IGFBP2 levels in PDAC patients (N = 75), chronic pancreatitis patients (N = 17), and healthy controls (N = 73).(a) The column bar graphs and error bars show mean ± SD.Serum IGFBP2 level was significantly elevated in PDAC.(b) Comparison of PDAC and ChPT patients and healthy controls on the basis of sex and age.

Figure 2 :
Figure 2: ROC operator characteristic curves for diagnosis of PDAC versus healthy controls and chronic pancreatitis.(a) ROC curves comparing PDAC patients to healthy controls for IGFBP2.(b) ROC curves comparing PDAC patients to ChPT patients for IGFBP2 and the combination of IGFBP2 plus CA19-9.AUC: area under the curve.

*
and**, P value for long-term diabetes or new onset diabetes versus no diabetes; *** and ****, P value for stage I/II versus stage IV and stage III versus stage IV.

Figure 3 :
Figure 3: Scatter plots for plasma IGFBP2 and CA19-9 levels in subgroups of PDAC patients.(a) IGFBP2 levels and (b) CA 19-9 levels in patients with and without lymph node involvement.(c) IGFBP2 levels and (d) CA 19-9 levels in patients with and without distant metastasis.(e) IGFBP2 levels and (f) CA 19-9 levels in patients with different diabetes status.(g) IGFBP2 levels and (h) CA 19-9 levels in patients at different tumor stages.

Figure 4 :
Figure 4: Association of plasma CA19-9 and IGFBP2 levels with survival time.Log Rank test was performed for curves comparison.The Kaplan-Meier survival curves for (a) PDAC patients grouped by the plasma CA19-9 cutoff value of 37 U/mL and (b) patients grouped by the plasma IGFBP2 cutoff value of 333.9 ng/mL.

Table 1 :
Basic clinicopathological information of patients

Table 2 :
Pancreatic tumor patient characteristics and plasma IGFBP2 levels (mean ± SD)

Table 3 :
Univariate and multivariate analyses for prognostic factors in PDAC cases (N = 75)* *Survival data were available for 75 patients; 8 were alive at the time of data analysis.